Abstract
BACKGROUND: Chronic alcohol use results in many pathological effects including alcoholic liver disease (ALD). ALD pathogenesis requires endotoxemia. Our previous studies showed that increased intestinal permeability is the major cause of endotoxemia, and that this gut leakiness is dependent on alcohol stimulation of inducible nitric oxide synthase (iNOS) in both alcoholic subjects and rodent models of alcoholic steatohepatitis. The mechanism of the alcohol-induced, iNOS-mediated disruption of the intestinal barrier function is not known. We have recently shown that alcohol stimulates activation of the transcription factor Snail and biomarkers of epithelial mesenchymal transition. As activated Snail disrupts tight junctional proteins, we hypothesized that activation of Snail by iNOS might be one of the key signaling pathways mediating alcohol-stimulated intestinal epithelial cell hyperpermeability. METHODS: We measured intestinal permeability in alcohol-fed C57BL/6 control and iNOS knockout (KO) mice, and measured Snail protein expression in the intestines of these mice. We then examined intestinal epithelial permeability using the Caco-2 cell model of the intestinal barrier ± small interfering RNA (siRNA) inhibition of Snail. We assessed Snail activation by alcohol in Caco-2 cells ± inhibition of iNOS with L-NIL or siRNA. Finally, we assessed Snail activation by alcohol ± inhibition with siRNA for p21-activated kinase (PAK1). RESULTS: Our data show that chronic alcohol feeding promotes intestinal hyperpermeability in wild-type BL/6, but not in iNOS KO mice. Snail protein expression was increased in the intestines of alcohol-treated wild-type mice, but not in iNOS KO mice. siRNA inhibition of Snail significantly inhibited alcohol-induced hyperpermeability in Caco-2 cell monolayers. Alcohol stimulation of Snail(pS246) activation was blocked by inhibition of iNOS with L-NIL or with siRNA. siRNA inhibition of PAK1 significantly inhibited alcohol-mediated activation of Snail in Caco-2 cells. CONCLUSIONS: Our data confirmed our prior results and further demonstrated that alcohol-induced gut leakiness in rodents and intestinal epithelial cell monolayers is iNOS dependent. Our data also support a novel role for Snail activation in alcohol-induced, iNOS-mediated intestinal hyperpermeability and that PAK1 is responsible for activation of Snail at Ser246 with alcohol stimulation. Identification of these mechanisms for alcohol-induced intestinal hyperpermeability may provide new therapeutic targets for prevention and treatment of alcohol-induced leaky gut, endotoxemia, and endotoxin-associated complications of alcoholism such as ALD.